Seasonal tissue-specific gene expression in wild crown-of-thorns starfish reveals reproductive and stress-related transcriptional systems

Animals are influenced by the season, yet we know little about the changes that occur in most species throughout the year. This is particularly true in tropical marine animals that experience relatively small annual temperature and daylight changes. Like many coral reef inhabitants, the crown-of-thorns starfish (COTS), well known as a notorious consumer of corals and destroyer of coral reefs, reproduces exclusively in the summer. By comparing gene expression in 7 somatic tissues procured from wild COTS sampled on the Great Barrier Reef, we identified more than 2,000 protein-coding genes that change significantly between summer and winter. COTS genes that appear to mediate conspecific communication, including both signalling factors released into the surrounding sea water and cell surface receptors, are up-regulated in external secretory and sensory tissues in the summer, often in a sex-specific manner. Sexually dimorphic gene expression appears to be underpinned by sex- and season-specific transcription factors (TFs) and gene regulatory programs. There are over 100 TFs that are seasonally expressed, 87% of which are significantly up-regulated in the summer. Six nuclear receptors are up-regulated in all tissues in the summer, suggesting that systemic seasonal changes are hormonally controlled, as in vertebrates. Unexpectedly, there is a suite of stress-related chaperone proteins and TFs, including HIFa, ATF3, C/EBP, CREB, and NF-κB, that are uniquely and widely co-expressed in gravid females. The up-regulation of these stress proteins in the summer suggests the demands of oogenesis in this highly fecund starfish affects protein stability and turnover in somatic cells. Together, these circannual changes in gene expression provide novel insights into seasonal changes in this coral reef pest and have the potential to identify vulnerabilities for targeted biocontrol.

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Summary
Wildtype animals in their native habitats, are often overlooked when considering experimental models.Most animals though, do not live in laboratories, housed under constant conditions, with ample food, conditioned light, and clean conditions.Biology is impacted by changes in the world, by reproduction, temperature change and physical conditions that constantly vary.The current dataset is a very nice demonstration of such dynamic changes in an important animal model.
The data are RNA-seq experiments of diXerent tissues from diXerent animals from diXerent times of the year.As such -it is exactly what investigators need in order to understand biology.These datasets will be very useful in looking at any further analysis of especially marine organisms.
Response: Thank you for your overall positive assessment of our approach and the manuscript.

Critique
It is unfortunate that the investigators did not include gonads in their analysis.Although gonads have been sequenced from this animal, they would serve as an excellent and essential foundation for measuring changes in other somatic tissues.Without the gonads, we are missing a key baseline for which all other tissues can be compared.It would serve as a seasonal control, especially since in this animal, the reproductive cycles are so well known.Further, the gonads may well be the regulators of all the other tissues sampled and we are left guessing what correlative changes we might find were those data also present.
Response: We would have liked to have included gonads in this study, however the mode of sampling (immediately upon collection on marine vessels at sea) and the sampling regime (small and precise tissue biopsies) precluded us procuring these in the winter, when the gonads were absent or reduced to an extent that they were not visually discernible from the underlying viscera.
Although we were not able to undertake a seasonal comparison of gonad gene expression, we previously have demonstrated that large di>erences exist between female and male gonads and somatic tissues prior to a spawning event (Jonsson et al. 2022. BMC Biol 20: 288).The seasonal analysis of the latter in the current manuscript has revealed sexual dimorphic gene regulation likely underlies di>erences in gene expression in fecund female and male COTS (new Fig 2D and S5 Table).
Further, as valuable as the datasets are -it would be important to have some supporting data to test RNA-values by e.g.qPCR, and to have in situ RNA hybridizations to see which cells of the various tissues the genes are actually expressed.These correlative experiments would help the reader better understand context for within the space that the data must sit.
Response: Thank you for these suggestions.Our sampling regime -multiple biological replicates for each somatic tissue in each season and sex (new Fig 1D,E, S2-4 Tables and https://doi.org/10.5281/zenodo.10831187)-provides a statistical perspective on gene expression that can not be readily achieved by in situ hybridization or quantitative RT-PCR analyses on individual starfish.Our dataset accounts for gene expression variation between individuals and seasons.We elected to emphasise genes that we deem significantly di>erentially expressed using Wald Test statistics in the DESeq2 package with an adjusted p-value < 0.05 between conditions (tissues, seasons and sex).This is a more rigourous approach compared to qRT-PCR, which is reliant on high levels of similarity of reference gene expression between individuals.Thus, we feel that both in situ hybridization and qRT-PCR will not further improve the conclusions drawn from the replicated transcriptomic data used in the current study.